The past decade has seen an explosion of interest in the role of epigenetics in cancer. Tumor suppressor gene silencing by promoter methylation is one of the key epigenetic mechanisms that contributes to tumorigenesis. The goal of the proposed application is to expand our understanding of the role of DNA methylation in tumor maintenance, by reducing DNA methylation in established colorectal tumors, using an innovative mouse model system in which the DNA methylation process can be reversibly and tightly inhibited through transcriptional repression of the endogenous Dnmt1 DNA methyltransferase gene. The first specific aim is to determine the role of DNA methylation in the maintenance of colorectal tumors. Our transcriptional repression technology enables us to circumvent cell lethality imposed by both conventional and conditional knock-out of Dnmt1, and allows us to produce the first mouse model with the capacity to conditionally and reversibly transcriptionally repress endogenous Dnmt1. The ability of our technology to suppress the DNA methylation process after the tumor has developed will allow tumors to develop under normal epigenetic influence, permitting an assessment of the role of DNA methylation in tumor maintenance without affecting tumor initiation. The inhibition of DNA methylation through transcriptional repression rather than the use of toxic compounds will allow a more specific assessment of the role of DNA methylation. The use of mouse cancer models for which the contribution of DNA methylation has been demonstrated will increase the likelihood of observing promising anti-neoplastic responses. The ability to temporally manipulate DNA methylation levels in tightly tissue-controlled context will provide unique abilities to investigate the role o epigenetics at defined time windows of tumor initiation, progression, invasion, and metastasis. The second specific aim is to comprehensively map aberrant DNA methylation changes involved in the colorectal tumor maintenance and development, and to identify candidate epigenetic driver events by comparison to human primary colorectal tumors. A combined genomics approach of whole genome shotgun bisulfite sequencing (WGSBS) and gene expression microarray analysis will allow us to efficiently map tumor- specific DNA methylation changes with an accompanying gene expression change. Comprehensive genome- wide DNA methylation data of 500 human primary colorectal cancer samples being produced in the lab in the context of TCGA will allow us to perform extensive validation of results obtained in our proposed mouse study. This proposed application will likely yield valuable insights into epigenetic contributions to the maintenance of established tumors, and will produce a genome-wide view of epigenetic alterations involved in the development and maintenance of colorectal tumors.